Review Near-Infrared Fluorescent Proteins: Multiplexing and Optogenetics across Scales 1 2 2,3 Daria M. Shcherbakova, Olesya V. Stepanenko, Konstantin K. Turoverov, and 1,4, Vladislav V. Verkhusha * Since mammalian tissue is relatively transparent to near-infrared (NIR) light, NIR Highlights fl fluorescentproteins(FPs) engineeredfrombacterialphytochromeshavebecome New monomeric NIR uorescent proteins (FPs)complement GFP-likeFPs forcross- widely used probes for non-invasive in vivo imaging. Recently, these genetically talk-free imaging. Spectrally distinct ver- encoded NIR probes have been substantially improved, enabling imaging experi- sions of these FPs are available, ranging from 670 to 720 nm in emission maxima. ments that were not possible previously. Here, we discuss the use of monomeric NIR FPs and NIR biosensors for multiplexed imaging with common visible GFP- The first NIR FRET biosensor, which based probes and blue light-activatable optogenetic tools. These NIR probes are detects Rac1 GTPase, is compatible with simultaneous imaging with CFP- suitable for visualization of functional activities from molecular to organismal YFP-based biosensors. levels. In combination with advanced imaging techniques, such as two-photon fl microscopy with adaptive optics, photoacoustic tomography and its recent Because they have excitation and uor- escence close to or within the NIR win- modification reversibly switchable photoacoustic computed tomography, NIR dow of tissue transparency (650– probes allow subcellular resolution at millimeter depths. 900 nm), NIR probes can be imaged across scales from subcellular to whole animals. Functional imaging is possible Monomeric NIR FPs and Biosensors Open New Possibilities for Imaging and using cell signaling, cell cycle, and Optogenetics protein–protein interaction reporters. NIR FPs derived from bacterial phytochrome photoreceptors(BphPs) (see Glossary) are Spectral properties of NIR FPs combined useful probes for imaging across spatial scales from subcellular to whole body. In microscopy, with advanced imaging approaches, NIR FPs allow spectral multiplexing with FPs of the GFP family and optogenetic tools activated such as structured illumination, two- by blue light. In in vivo imaging, deep penetration of NIR light in tissue, low autofluorescence, photon microscopy, and photoacoustic tomography, enable subcellular resolu- and reduced scattering in NIR make NIR FPs superior to GFP-like FPs (reviewed in [1]). NIR FPs tion at millimeter depths. have been widely used in various fields of biology and biomedicine, including cancer research 1 [2–7], neuroscience [8–10], stem cell studies [11–13], parasitology [14], and virology [15], Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics wherein the probes’ NIR spectra allow their non-invasive visualization in vivo or labeling of Center, Albert Einstein College of organelles and cells for multicolor imaging. Medicine, Bronx, NY 10461, USA 2 Laboratory of Structural Dynamics, Stability and Folding of Proteins, Most NIR FPs are engineered that bind the PAS and GAF domains of BphPs. As chromo- Institute of Cytology, Russian phores, bacterial phytochromes use biliverdin (BV), which is available in mammalian cells as a Academy of Sciences, St. Petersburg product of heme breakdown. Currently, there are about 20 different NIR FPs developed from 194064, Russian Federation 3 Department of Biophysics, Peter the bacterial phytochromes that can be grouped in series named by their developers: iRFPs [16,17] Great St. Petersburg Polytechnic and miRFPs [18,19], IFPs [20,21] and mIFP [22], and Wi-Phy [23]. The m/iRFP and m/IFP University, St. Petersburg 195251, series, which were tested for their performance in mammalian cells, differ in their molecular Russian Federation 4 Department of Biochemistry and brightness, effective brightness when expressed in mammalian cells (cellular brightness), Developmental Biology, Faculty of spectral properties, and oligomeric state (Table 1, Key Table). A recent NIR FP developed from Medicine, University of Helsinki, cyanobacterial phycobiliprotein photoreceptor smURFP [24] is considerably dimmer than Helsinki 00290, Finland *Correspondence: iRFPs [25], although it is an interesting engineering result showing that a photoreceptor [email protected] may be evolved to bind BV autocatalytically. (V.V. Verkhusha). 1230 Trends in Biotechnology, December 2018, Vol. 36, No. 12 https://doi.org/10.1016/j.tibtech.2018.06.011 © 2018 Elsevier Ltd. All rights reserved. Key Table Table 1. BphP-Derived NIR FPs with Demonstrated In Vivo and Live-Cell Applications and Far-Red GFP-like FPs NIR FP Excitation Emission Extinction coeffi- Quantum Molecular Molecular Oligomeric state Photostability in pKa Brightness in HeLa Refs À À (nm) (nm) cient (M 1cm 1) yield (%) brightnessa brightness mammalian cells versus b versus cells, t1/2 (s) iRFP713 (%) iRFP713 (%) miRFP670 642 670 87 400 14.0 12.2 198 Monomer 490 (155) 4.5 72 [18] miRFP703 674 703 90 900 8.6 7.8 127 650 (394) 4.5 37 miRFP709 683 709 78 400 5.4 4.2 69 500 (192) 4.5 30 mIFPb,c 683 (683) 705 (704) 65 900 (82 000) 6.9 (8.4) 4.6 74 90 (54) 4.5 15 [22] IFP2.0b,c 688 (690) 709 (711) 72 900 (98 000) 6.8 (8.1) 5.0 80 Dimerd 150 (108) 4.5 8 [21,22] iRFP670 643 670 114 000 12.2 13.9 225 Dimer 290 4.5 119 [17,21] iRFP682 663 682 90 000 11.1 10.0 162 490 4.5 105 iRFP702 673 702 93 000 8.2 7.6 124 630 4.5 61 iRFP713 (aka iRFP) 690 713 98 000 6.3 6.2 100 960 4.5 100 [16] iRFP720 702 720 96 000 6.0 5.8 93 490 4.5 112 [16,17] miRFP670-2 643 670 103 000 13.6 14.0 227 Monomer 310 4.5 72 [29] miRFP682 663 682 91 000 11.2 10.2 165 500 4.5 117 miRFP702 673 702 88 000 8.1 7.1 115 640 4.5 37 miRFP713 690 713 99 000 7.0 6.9 112 980 4.5 109 miRFP720 702 720 98 000 6.1 6.0 97 510 4.5 116 [19] e f Trends mCherry 587 610 72 000 22.0 15.8 255 Monomer NA 3.8 NA [19,28] FusionRede 580 608 83 000 19.0 15.8 255 NA 4.6 NA [28,37] in e g Biotechnology, mNeptune 600 650 57 500 20.0 11.5 185 Dimer NA 5.4 NA [37,72,73] mCardinale 603 651 79 000 18.0 14.2 229 NA 5.3 NA [72–74] À À aDetermined as a product of extinction coefficient at excitation maximum (in mM 1cm 1) and fluorescence quantum yield. b December Determined as an effective NIR fluorescence in transiently transfected live HeLa cells with no supply of exogenous BV and after normalization to fluorescence of co-transfected EGFP and overlap of FP spectra with excitation laser and emission filters. cCharacteristics of NIR FPs shown in original publications are in parentheses. dOriginally reported as a monomer, IFP2.0 was later found to be a dimer [22]. 2018, eFar-red GFP-like FP. fNA, not available. Vol. gOriginally reported as monomers, mNeptune and mCardinal were later found to be dimers [73]. 36, No. 12 1231 Relatively low quantum yield of NIR FPs is compensated by their high extinction coefficient. Glossary The resulting molecular brightness of NIR FPs is comparable to modern far-red FPs of the Adaptive optics (AO): correction of GFP family (Table 1). NIR FPs are used in microscopy in the same constructs and imaging aberrations in scattering tissue to enhance resolution of the optical conditions as GFP-like probes. However, it is important to use light sources that produce imaging at depth. A direct wavefront- adequate light intensities in NIR, such as xenon lamps. In addition, cellular and tissue sensing approach using fluorescence fl auto uorescence is lowest in the NIR spectral region [4,26]. In deep-tissue imaging, iRFPs guide stars is a method to correct were shown to be superior to the brightest available far-red GFP-like FPs in direct these aberrations and increase depth of high-resolution imaging. comparison [16,17], due to low autofluorescence in NIR combined with better NIR light All-optical electrophysiology: an penetration and less scattering. Interestingly, the relatively dim and the most red-shifted approach that combines light- iRFP720 was found to be the most sensitive probe among FPs for deep-tissue imaging induced perturbation (using [15,27]. channelrhodopsins as optogenetic tools) and optical readout (via genetically encoded sensors for Apparent brightness of NIR FPs in cells, so-called cellular brightness, does not always membrane voltage or calcium) of correlate with molecular brightness [1,25]. It also depends on protein stability and BV binding neuronal activity. Bacterial phytochrome efficiency. Early NIR FPs, such as IFP1.4 [20], suffered from poor BV incorporation in photoreceptors (BphPs): mammalian cells, leading to their poor brightness (8% for IFP1.4 versus iRFP713 [17]). multidomain photoreceptors that Screening protein variants in mammalian cells during protein engineering allowed iRFPs to respond to near-infrared (NIR) light be obtained that do not require an exogenous BV supply and that can be used similarly to and incorporate the most red-shifted natural chromophore biliverdin (BV). GFP-like probes [16,17]. They participate in microbe light- adaptive behavior and activate a Bright NIR FPs, such as those in the iRFP family, are dimers and may not be suitable for biochemical response after light- protein tagging and engineering of biosensors as they can interfere with proper localization induced chromophore isomerization and conformational changes in the and function of fused functional molecules by inducing their dimerization. The recent devel- protein molecule. opment of bright monomeric NIR FPs [18,19,22] has changed this situation the same way that Bimolecular fluorescence the development of monomeric mFruits [28] from dimeric DsRed GFP-like FPs did a decade complementation (BiFC): an – ago.